In general, prior art fluorometers suffer from a common problem of being unable to discriminate between the generated fluorescent signal and the background noise. Certain types of conventional fluorometers discriminate between the fluorescent signal and the background noise on the basis of wavelength. This type of discrimination is generally not sufficient for many types of fluorescent signals.
Another type of discrimination can be accomplished using a time-gaged technique. In particular, these instruments are based on the principle of permitting the observation of the fluorescence or luminescence a short, and if desired a variable, time after the excitation period. Time gaged fluorometers therefore add an additional level of discrimination by viewing the signal fluorescence during an optimal time window. In the past, this technique generally employed a fluorophore of long decay time in order to allow the background fluorescence to decay. The long decay times produced relatively slow measurements and lost information afforded by the polarization. Furthermore, the measurements were clouded because they contained a considerable amount of background noise.
The time gaged technique is in general based on a phosphoroscope invented by Becquerel in 1867. In the Becquerel instrument, the luminescent substance is placed between two rotating discs which are mounted on a common axis and which have sector shaped apertures. The variable time gaging is achieved by an adjustment of the angle between a sector on one disc and a sector on the other. Subsequent refinements of the time gaging technique have been accomplished by the use of spark discharges, oscilloscopes, Kerr cells, supersonic cells and lasers.
The rotating disc invented by Becquerel was put into a conical configuration for a microscope by Jones as described in U.S. Pat. No. 2,071,408 in 1937. Other more recent improvements have used electronic techniques. For example, U.S. Pat. No. 4,341,957 to Wieder provided for the gaging of a detecting circuit electronically and used a laser for excitation. In this way, as in other refinements of the Becquerel phosphoroscope, the gaging mechanism may be adjusted so that observation of the desired signal can be optimized within the limits of the phosphoroscope. Other prior art devices such as U.S. Pat. No. 4,006,360 to Mueller use electronic gaging to distinguish between species of differing decay times where two species are involved and one is a bound dye and the other is an unbound dye.
Several commercial instruments are currently available for the measurement of decay times or lifetimes. These instruments utilize nanosecond flash sources (electric spark in air at reduced pressure). One instrument introduces the output of a photomultiplier tube to a fast oscilloscope. Provision is made to match the experimental curve with a sum to 3 or 4 exponentials.
A second instrument excites the sample by repeated flashes from the source (such as at 5 kHz) and pulses the photomultiplier at progressively increased times after each flash. The output is fed into a recorder or computer to provide an intensity vs. time signal. In addition, this instrument is supplied with software to reconvolute the experimental curve by a well known method termed Linearized Least Squares Reconvolution.
A third instrument utilizes a flash source (typically at 1 kHz). A timer is started at the time of the flash and stopped when the photomultiplier detects a photon from the sample.
Subsequently, counts are accumulated in a series of time boxes giving a relationship leading to the decay curve for the fluorescence.
Both wavelength-based discrimination and time-based discrimination suffer by having background fluorescence superimposed on the signal with only an indirect means of segregating the background fluorescence from the signal. In addition, the use of dyes of long decay time effectively smears the desired signal over a long time period, thus making this signal hard to extract. Dyes of long decay time have inherently low extinction coefficients and therefore provide inefficient excitation of the fluorescence.
In co-pending application Ser. No. 751,746, now U.S. Pat. No. 4,877,9 filed and assigned of record to the assignee of record by Walter B. Dandliker on Jul. 1, 1985, for a "Fluorometer" of this application, a new type of fluorometer is disclosed and claimed which permits the signal from the fluorophore to be automatically separated from the background in an improved manner to produce an enhanced fluorescent signal. This enhancement of the fluorescent signal occurs by a particular instrument design, by the type of data collected and by a specific method used to process this data.
In co-pending application Ser. No. 751,746, now U.S. Pat. No. 4877965, a source such as a laser is pulsed to produce concentrated light energy at a particular frequency such as 780 nm. This light is directed to a specimen to obtain a fluorescence from particular fluorophore molecules in the specimen. This fluorescence continues for a period of time after the initiation of the fluorescence. A detector is provided to detect the fluorescence from the particular fluorophore molecules in the specimen. The detector is gaged to operate only for a particular period of time beginning after the initiation of the fluorescence of the particular fluorophore molecules in the specimen and ending before the completion of the fluorescence of the particular fluorophore molecules in the specimen. By gaging the detector in this manner, the signals produced from the fluorescence of the particular fluorophore molecules in the detector are enhanced relative to the signals representing background noise and the time dependence of these signals provides the information for a further discrimination between desired signal and background.